Pharmaceutical compound capable of induce immune protective response against Dengue virus having the capsid protein of the Dengue virus

ABSTRACT

This invention describes a pharmaceutical compound which has the capsid protein of Dengue virus and which is capable of inducing, in the receptor, a protective immune response before the viral challenge, without inducing the Ab-dependent enhancement phenomenon.

This application is the U.S. National Phase of, and Applicants claimpriority from, International Application Number PCT/CU2006/000008 filed18 Sep. 2006 and Cuban Application bearing Serial No. CU2005-0168 filed16 Sep. 2005, which are incorporated herein by reference.

The present invention is related to the field of biotechnology and thepharmaceutical industry, in particular to the obtaining of proteinscapable of inducing an immune response against the infection with Denguevirus, quoted from now on as DEN, while avoiding the antibody-dependentenhancement phenomenon that has been described in persons re-infectedwith this virus.

Dengue fever (DF) and dengue hemorrhagic fever (DHF) acquire increasingimportance as health problems, affecting several countries of thetropical and subtropical zones of the planet. Dengue virus has beenrecognized in more than 100 countries and 2 500 million people living inrisk areas are estimated. Between 50 and 100 million cases from DF and250 000 to 500 000 of DHF are reported each year. (Guzmán M. G. andKouri G. 2002. Dengue: an update. Lancet Infect. Dis. 2: 33-42).

The causal agent of this disease is the Dengue virus of the genusFlavivirus, family Flaviviridae, which is transmitted by the mosquitoAedes aegypti (Leyssen P., De Clerco E., Neyts J. 2000. Perspectives forthe treatment of infections with Flaviviridae. Clin. Microbiol. Rev. 13:67-82).

Until now four serotypes have been reported that can circulate in a sameregion. Dengue virus is an RNA positive coated virus, whose genomecontains only one reading frame. This RNA is translated in a polyproteinthat is processed in three structural proteins and seven non-structuralproteins. (Russell P. K., Brandt W. E., Dalrymple J. M. 1980. Chemicaland antigenic structure of flaviviruses. The togaviruses: biology,structure, replication. Schelesinger R. W. (ed.). 503-529).

Multiple epidemiological studies have been made to determine the riskfactors that entail to the most severe form of Dengue disease. This ischaracterized by high fever, extrusion of liquids, hemorrhages andfinally the Dengue shock. (Gubler D. J. 1998. Dengue and DengueHemorrhagic Fever. Clin. Microbiol. Rev. 11: 480-496). One of the mostimportant risk factors is the secondary infection by a heterologousserotype. Cross-protection among the infections of the differentserotypes does not exist. (Kouri G., Guzman M. G., Bravo J., Trina C.1989. Dengue hemorrhagic fever/dengue shock syndrome: lessons from theCuban epidemic. WHO Bulletin OMS. 67: 375-380).

Several hypotheses exist to explain this phenomenon. One of the mostimportant is the antibody depend enhancement. (Halstead S. B., ScanlonJ. E., Umpaivit P., Udomsakdi S. 1969. Dengue and Chikungunya virusinfection in man in Thailand, 1962-1964. IV. Epidemiologic studies inthe Bangkok metropolitan area. Am. J. Trop. Med. Hyg. 18: 997-1021).

From the first studies, it was raised that DEN virus replicates ingreater measurement in peripheral mononuclear cells from the blood ofpatients who had undergone a previous infection with the virus (HalsteadS. B., O'Rourke E. J., Allison A. C. 1977. Dengue viruses andmononuclear phagocytes. II. Identity of blood and tissue leukocytessupporting in vitro infection. J. Exp. Med. 146: 218-229). Later, it wasdemonstrated that the residual antibodies were the responsables of thiseffect (Morens D M, Halstead S B, Marchette N J. 1987. Profiles ofantibody-dependent enhancement of dengue virus type 2 infection. MicrobPathog. October; 3(4):231-7).

In conditions of specificity or concentration of antibodies in whichthere is not neutralization, the antibody-virus complexes can beinternalized by cells presenting Fey receptors in the membranes, likemonocytes and macrophages. This mechanism, known as antibodies-dependentenhancement (ADE) occurs during secondary infections. (Morens D M,Halstead S B, Marchette N J. 1987. Profiles of antibody-dependentenhancement of dengue virus type 2 infection. Microb Pathog. October;3(4):231-7; Kliks S. C., Nimmannitya S., Nisalak A., Burke D. S. 1988.Evidence that maternal dengue antibodies are important in thedevelopment of dengue hemorrhagic fever in infants. Am. J. Trop. Med.Hyg. 38: 411-419).

Halstead et al. (Halstead S. B., Scanlon J. E., Umpaivit P., UdomsakdiS. 1969. Dengue and Chikungunya virus infection in man in Thailand,1962-1964. IV. Epidemiologic studies in the Bangkok metropolitan area.Am. J. Trop. Med. Hyg. 18: 997-1021.), in a 3-year study in Bangkok,Thailand, reported that the hospitalization indices by DEN infectionamong children, reached a maximum in those between 7 and 8 months old.These indices were four to eight times greater than the observed betweenchildren of 1-3 months and twice than that in children of 3 years. Klikset al. (Kliks S. C., Nimmannitya S., Nisalak A., Burke D. S. 1988.Evidence that maternal dengue antibodies are important in thedevelopment of dengue hemorrhagic fever in infants. Am. J. Trop. Med.Hyg. 38: 411-419), determined the relation between the maternalneutralizing antibody titers against DEN-2 and the ages of thirteenchildren with FHD caused by the infection with the homologous virus. Theresults showed that the infection serious cases with the virus occurredwhen the maternal antibody levels diminished to sub-neutralizing levels.These data are consistent with the hypothesis in which the maternalantibodies play the double role of protecting first and stimulate thedevelopment of DHF later on.

Despite this immunological phenomenon, nowadays the most advancedvaccine candidates worldwide are based in attenuated virus of thedifferent four serotypes, containing the envelope protein. Thesecandidates are able to induce potential amplifying antibodies againstthe exposed proteins (PrM/M and Envelope) and protector neutralizingantibodies against the four viral serotypes in human volunteers.(Kanesa-thasan N., Sun W., Kim-Ahn G., Van Albert S., Putnak J. R., KingA., Raengsakulsrach B., Christ-Schmidt H., Gilson K., Zahradnik J. M.,Vaughn D. W., Innis B. L., Saluzzo J. F. y Hoke C. H. 2001. Safety andimmunogenicity of attenuated dengue virus vaccines (Aventis Pasteur) inhuman volunteers. Vaccine. 19: 3179-3188).

High levels of neutralizing antibodies after immunization could preventthe viral replication despite the induction of enhancing antibodies. Theproblem can take place when the total seroconversion to the fourserotypes in the vaccines, in terms of neutralizing Abs, is not obtainedor is diminished to low levels in blood and the individuals would becomethen susceptible to a severe secondary infection with a viral serotypewhose protective antibodies are not present. In fact, several tests inmonkeys and humans have been made to define the viral amounts in thevaccine formulations. (Guirakhoo F., Arroyo J., Pugachev K. V., MillerC., Zhang Z.-X., Weltzin R., Georgakopoulos K., Catalan J., Ocran S.,Soike K., Raterree M., Monath T. P. 2001. Construction, safety, andimmunogenicity in nonhuman primates of a chimeric yellow fever-denguevirus tetravalent vaccine. J. Virol. 75: 7290-7304).

In some cases the balance of seroconversion has not been obtained to thefour serotypes (Sabchareon A, Lang J, Chanthavanich P, Yoksan S, ForratR, Attanath P, Sirivichayakul C, Pengsaa K, Pojjaroen-Anant C,Chokejindachai W, Jagsudec A, Saluzzo J F, Bhamarapravati N. 2002.Safety and immunogenicity of tetravalent live-attenuated dengue vaccinesin Thai adult volunteers: role of serotype concentration, ratio, andmultiple doses. Am J Trop Med Hyg. 66(3): 264-72). In addition, it hasbeen necessary to administer up to three doses of attenuated vaccines inchildren for a total seroconversion, in terms of neutralizing antibodiesand still not known whether these will last in the time (Sabchareon A,Lang J, Chanthavanich P, Yoksan S, Forrat R, Attanath P, SirivichayakulC, Pengsaa K, Pojjaroen-Anant C, Chambonneau L, Saluzzo J F,Bhamarapravati N. 2004. Safety and immunogenicity of a three doseregimen of two tetravalent live-attenuated dengue vaccines in five- totwelve-year-old Thai children. Pediatr Infect Dis J.; 23(2):99-109).This is one of the most questionable concerns in the vaccineformulations that include the envelope protein of Dengue virus,consequently of the vaccines candidates in development.

Another drawback of the attenuated vaccines, currently in phase I/II, isthe safety. Upon the first dose the existence of adverse effects inadults and children like fever, myalgia, petequias and headache, hasbeen demonstrated in several studies (Sabchareon A, Lang J,Chanthavanich P, Yoksan S, Forrat R, Attanath P, Sirivichayakul C,Pengsaa K, Pojjaroen-Anant C, Chambonneau L, Saluzzo J F, BhamarapravatiN. 2004. Safety and immunogenicity of a three dose regimen of twotetravalent live-attenuated dengue vaccines in five- to twelve-year-oldThai children. Pediatr Infect Dis J.; 23(2):99-109). In general thephenomenon of reversion to the virulence potentially associated to livevaccines may be presented.

In the search of new alternatives, it has been developed differentvariants of vaccine candidates based on the envelope protein orfragments of this one, obtained through a recombinant way. Theserecombinant candidates avoid the safety problems related to theinoculation of live virus, and are able to sensitize the individual if abalanced response against the four serotypes is not induced (Velzing J,Groen J, Drouet M T, van Amerongen G, Copra C, Osterhaus A D, Deubel V.1999. Induction of protective immunity against Dengue virus type 2:comparison of candidate live attenuated and recombinant vaccines.Vaccine. March 17; 17(11-12):1312-20). On the other hand, thesecandidates require powerful adjuvants—not approved for their use inhumans yet—to stimulate a serotype-specific suitable protective immuneresponse (Hermida L, Rodriguez R, Lazo L, Silva R, Zulueta A, Chinea G,Lopez C, Guzman M G, Guillen G. 2004. A dengue-2 Envelope fragmentinserted within the structure of the P64k meningococcal protein carrierenables a functional immune response against the virus in mice. J VirolMethods. 2004 January; 115(1):41-9).

The humoral response of neutralizing antibodies has been extensivelystudied in animals and its protector effect has been demonstrated. Thecytotoxic cellular immune response as a protector mechanism in Denguehas not deeply been explored. On the contrary, there are several reportsin which the correlation between the induction of a cellular responseand the most severe form of the disease is demonstrated (Rothman A. L. yEnnis F. A. 1999. Immunopathogenesis of Dengue Hemorragic Fever.Virology. 257: 1-6). These studies are based on the presence of highlevels of activated T-cells in those individuals that exhibit DHF (GreenS, Pichyangkul S, Vaughn D W, Kalayanarooj S, Nimmannitya S, Nisalak A,Kurane I. Rothman A L, Ennis F A. 1999. Early CD69 expression onperipheral blood lymphocytes from children with dengue hemorrhagicfever. J Infect Dis. 180(5):1429-35).

T-cells epitopes have been reported mainly in nonstructural proteins(Kurane I, Zeng L, Brinton M A, Ennis F A. 1998. Definition of anepitope on NS3 recognized by human CD4+ cytotoxic T lymphocyte clonescross-reactive for dengue virus types 2, 3, and 4. Virology. 1998 Jan.20; 240(2):169-74), but also are present in the envelope and in thecapsid proteins (Bukowski, J. F., I. Kurane, C.-J. Lai, M. Bray, B.Falgout, and F. A. Ennis. 1989. Dengue virus-specific cross-reactive CD8human cytotoxic T lymphocytes. J. Virol. 63:5086-5091; Gagnon S. J.,Zeng W., Kurane I., Ennis F. A. 1996. Identification of two epitopes onthe dengue 4 virus capsid protein recognized by a serotype-specific anda panel of serotype-cross-reactive human CD4+ cytotoxic T-lymphocyteclones. J Virol. 70: 141-147). Nevertheless, the protective character ofsome of these proteins based only on the induction of a cellular immuneresponse has not been demonstrated.

In the search of vaccine candidates that avoid the immunoenhancementphenomenon. studies using the non-structural proteins NS1 and NS3 havebeen made. In the case of NS1, some level of protection in miceimmunized with the recombinant protein has been reached.

Similar results have been obtained using a naked DNA containing the NS1gene, through the ADCC mechanism (Wu S F. Liao C L, Lin Y L, Yeh C T,Chen L K, Huang Y F, Chou H Y, Huang J L, Shaio M F, Sytwu H K. 2003.Evaluation of protective efficacy and immune mechanisms of using anon-structural protein NS1 in DNA vaccine against dengue 2 virus inmice. Vaccine. September 8; 21(25-26):3919-29). Nevertheless, reports oftheir possible roll in phenomena of autoimmunity due to the induction ofantibodies that recognize human endothelial cells exist (Chiou-Feng Lin,Huan-Yao Lei, Ai-Li Shiau, Hsiao-Sheng Liu, Trai-Ming Yeh, Shun-HuaChen, Ching-Chuan Liu, Shu-Chen Chiu, and Yee-Shin Lin. 2002.Endothelial Cell Apoptosis Induced by Antibodies against Dengue Virusnonstructural Protein 1 via Production of Nitric Oxide. J. Immunol.657-664). Additionally, there is a report of protection using a nakedDNA formulation containing the NS3 gene; however, it was demonstratedthat this protection was mediated by the raised antibodies since thesame protection using passive immunization was obtained (Tan C H, Yap EH, Singh M, Deubel V, Chan Y C. 1990. Passive protection studies in micewith monoclonal antibodies directed against the non-structural proteinNS3 of dengue 1 virus. J Gen Virol. 1990 March; 71 (Pt 3):745-9). Inaddition, it is worth noting the hypothesis, that the cellular responsecan be potentially harmful facing an infection with heterologous virusbased on studies of NS3 protein epitopes (Zivny J, DeFronzo M, Jarry W,Jameson J, Cruz J, Ennis F A, Rothman A L. 1999. Partial agonist effectinfluences the CTL response to a heterologous dengue virus serotype. JImmunol. September 1; 163(5):2754-60).

In the case of the capsid protein of dengue virus, no evidences ofprotection in the challenge with a lethal dengue virus are reported.Concerning related flaviviruses, a report was published where authorsinoculated mice with a naked DNA formulation containing the gene of theJapanese Encephalitis (JE) capsid protein. This formulation did notinduce a protective response against the challenge with lethal JE inmice, despite the demonstration of a cytotoxic response (Konishi E,Ajiro N, Nukuzuma C, Mason P W, Kurane I. 2003. Comparison of protectiveefficacies of plasmid DNAs encoding Japanese encephalitis virus proteinsthat induce neutralizing antibody or cytotoxic T lymphocytes in mice.Vaccine. September 8; 21(25-26):3675-83).

Protection using the recombinant protein capsid has been demonstratedonly in the case of human papilloma virus. However it has been suggestedits protector role with other virus like Hepatitis C virus.Nevertheless, in all cases, they are chronic infections or tumors, inwhich the cellular cytotoxic response is the only mean of the immunesystem to clear viral infection (Duenas-Carrera S, Alvarez-Lajonchere L,Alvarcz-Obregon J C, Herrera A, Lorenzo L J, Pichardo D, Morales J.2000. A truncated variant of the hepatitis C virus core induces a slowbut potent immune response in mice following DNA immunization. Vaccine.November 22; 19(7-8):992-7; Suzich J A, Chin S J, Palmer-Hill F J, etal. 1995. Systemic immunization with papillomavirus L1 proteincompletely prevents the development of viral mucosal papillomas. ProcNatl Acad Sci USA; 92: 11553-57). These diseases do not correspond withthe acute profile that is exhibited in the infection by Dengue in humans(Vaughn D. W., Green S., Kalayanarooj S., Innis B. L., Nimmannitya S.,Suntayakorn S., Endy T. P., Raengsakulrach B., Rothman A. L., Ennis F.A. y Nisalak A. 2000. Dengue viremia titer, antibody response pattern,and virus serotype correlate with disease severity. J Infect Dis. 181:2-9).

The capsid protein of Dengue virus has a molecular weight of 9 to 12 kDa(112-127 amino acids) and has a marked basic character because the 25%of its amino acids are arginine and lysine. The presence of these aminoacids could favor antigenic presentations to the immune system due tothe capacity of polycationic peptides to do so. (Lingnau K., Egyed A.,Schellack C., Mattner F, Buschle M., Schmidt W. 2002. Poly-1-argininesynergizes with oligodeoxynucleotides containing CpG-motifs (CpG-ODN)for enhanced and prolonged immune responses and prevents theCpG-ODN-induced systemic release of pro-inflammatory cytokines. Vaccine.20: 3498-3508). The protein is located totally within the virionstructure without any exposed region (Kuhn R J, Zhang W, Rossmann M G,Pletnev S V, Corver J, Lenches E, Jones C T, Mukhopadhyay S, Chipman PR, Strauss E G, Baker T S, Strauss J H. 2002. Structure of dengue virus:implications for flavivirus organization, maturation, and fusion. Cell.March 8: 108(5):717-25).

Jones y cols. (Christopher T. Jones, Lixin Ma, John W. Burgner, TeresaD. Groesch, Carol B. Post, and Richard J. Kuhn. 2003. Flavivirus CapsidIs a Dimeric Alpha-Helical Protein. Journal of Virology, p 7143-7149,Vol. 77, No. 12) purified the capsid protein of VD2 obtained by therecombinant way in Escherichia coli (E. coli) and demonstrated that thisprotein behaves like a dimmer in solution without nucleic acids. Itssecondary structure is mainly in form of alpha-helices and is composedby four of these helices, being one of those of greater length in theC-terminal end. The N-terminal end does not present a defined structureand its deletion does not affect the structural integrity of theprotein.

This invention describes for the first time that the capsid of DEN-2virus, obtained by a recombinant way in E. coli and with only a 40% ofpurity, is able to induce a protective immune response against thechallenge with lethal DEN-2 virus in mice. It was demonstrated that thishighly purified protein, retained its protective capacity, which wassurpassed in the immunization of mice with the particulated form of themolecule. Moreover, it was demonstrated that the reached protection wasmediated by CD8+ T-cells, a novel element considering that the reportedT-cells epitopes for the capsid so far, are recognized by CD4+ T cells(Gagnon S J, Zeng W, Kurane I, Ennis F A. 1996. Identification of twoepitopes on the dengue 4 virus capsid protein recognized by aserotype-specific and a panel of serotype-cross-reactive human CD4+cytotoxic T-lymphocyte clones. J Virol. 70(1): 141-7; Simmons C P, DongT, Chau N V, Dung N T, Chau T N, Thao le T T, Dung N T, Hien T T,Rowland-Jones S, Farrar J. 2005. Early T-cell responses to dengue virusepitopes in Vietnamese adults with secondary dengue virus infections. JVirol. 79(9):5665-75). Additionally, this recombinant molecule was mixedwith the PD5 protein, which is formed by the P64k protein of Neisseriameningitidis and the III domain of the envelope protein of the Dengue-2virus. This fusion protein is able to generate a highly serotypespecific, protective and neutralizing immune response, with a lowprobability of generating the phenomenon of antibodies dependentenhancement (Hermida L, Rodriguez R, Lazo L, Silva R, Zulueta A, ChineaG, Lopez C, Guzman M G, Guillen G. 2004. A dengue-2 Envelope fragmentinserted within the structure of the P64k meningococcal protein carrierenables a functional immune response against the virus in mice. J VirolMethods. 2004 January; 115(1):41-9).

The obtaining of a genetic construction formed by the fusion of thecapsid protein and the III domain of the envelope protein is alsodescribed to reach the same objective. As a result, the two formulationswhere the capsid is combined with the III domain of DEN-2, generated alymphoproliferative response in mice higher than that generated by thecapsid only, and in addition a serotype specific antibodies responsehigher than the generated by PD5 only. This last result demonstrates theimmunoenhancing capacity of the capsid protein of dengue virus in thegeneration of Abs by a heterologous antigen, phenomenon described forother recombinant capsids from other viruses like the hepatitis B virus(Alvarez J C, Guillén G. Formulations containing virus like particles asimmunoenhancers by mucosal route. Cuban office of the Industrialproperty. CU 1998/183).

DETAILED DESCRIPTION OF THE INVENTION

The objective of this invention is to obtain a recombinant proteincorresponding to the capsid protein of Dengue virus, which generates aprotective response against the infection with the lethal virus when isinoculated in mice.

The gene codifying for the capsid protein of Dengue virus was insertedinto a plasmid containing the phage T5 promoter. The cells XL-1Blue,transformed with the recombinant plasmid, expressed high levels of theresulting protein.

This protein was purified approximately till a 40% of purity, and wasadjuvated in aluminum hydroxide to be inoculated in Balb/c mice. A monthupon the last dose the antiviral antibody response was measured. At thesame time the lymphoproliferative response in spleens stimulated invitro with the dengue virus was determined. As a result no antiviralantibodies were induced while a significant lymphoproliferative responsewas detected. In parallel, in not bleeding mice, the protection assaywas done. A lethal doses corresponding to 100 LD₅₀ of Dengue virus wasinoculated, the disease symptoms and death were observed during 21 days.As a result a 44% of survival-immunized mice were obtained while in thenegative control group all mice died. This is the first evidence of aprotective response against Dengue virus by the immunization only withthe capsid protein.

Later, a high-resolution purification process was conducted, obtaining a95% of purity of the recombinant protein.

Both preparations, the semi- and purified ones, were analyzed by HPLC toknow the aggregation state of the protein in each sample. In thesemipurified preparation was detected a fraction with lower retentiontimes, while in the purified sample a retention time corresponding tothe dimeric form of the molecule was detected.

To obtain an aggregation state in the purified variant, an in vitroparticulation process employing low quantities of oligonucleotides wasdone. As a result of the process, particles of 21 nm of diameter wereobtained.

The dimeric and particulated preparations, both with more than 95% ofpurity, were inoculated in mice. The dimeric preparation was adjuvatedwith Freund Adjuvant and aluminum hydroxide, while the particulatedvariant was adjuvated only with aluminum hydroxide.

Similar to the semipurified preparation, high levels oflymphoproliferation were detected. In the protection assay a 40 and 20%of survival were obtained with the dimeric preparation adjuvated withFreund and aluminum, respectively; however, the particulated proteinadjuvated with aluminum induced a higher protection percentage.

These results together with those obtained with the semipurified proteinshowed the capacity of the capsid protein of inducing a protectiveresponse in Balb/c mice and demonstrated the superiority of theparticulated form of the protein, letting it to be used to humans in thefuture together to the aluminum hydroxide as adjuvant. Additionally, notinducing an antiviral response would eliminate the phenomenon ofantibodies dependent enhancement as a risk factor for the occurrence ofthe most severe form of the disease: the dengue hemorrhagic fever.

With the aim to determine the possible mechanism of protection, which itis not related to the induction of Abs due to its demonstrated absence,a study of CD8+ cells depletion was made. As a result, the protectionreached with pure proteins of each variant was dependent of the presenceof the cells that present this marker, since eliminating them theinduced protective effect disappear.

Similarly, a study was made to know if the combination of theparticulated recombinant capsid with antigens inducing humoral responsedoes not affect the generation of the lymphoproliferative response andto count with a mixture of immunogens able to contribute to bothbranches of the immune response. To this end, the purified particulatedvariant of the capsid and a fusion protein containing the III domain ofthe envelope protein of the dengue-2 virus was inoculated in mice, whichis able to generate a serotype-specific immune response diminishing thephenomenon of ADE (Hermida L, Rodriguez R, Lazo L, Silva R, Zulueta A,Chinea G, Lopez C, Guzman M G, Guillen G. 2004. A dengue-2 Envelopefragment inserted within the structure of the P64k meningococcal proteincarrier enables a functional immune response against the virus in mice.J Virol Methods. 2004 January; 115(1):41-9). When administering threedoses and analyzing the raised Abs, it was demonstrated a higherinduction of antiviral serotype specifics Abs. As well, alymphoproliferative response higher than that induced only by the capsidand significantly higher than that induced by the fusion protein wasdetected.

In parallel, to know whether it is possible to obtain the combinationeffect using a genetic fusion of both antigens, a plasmid containing theIII domain of the envelope protein of DEN-2 virus fused to theN-terminal of the capsid protein gene was constructed. The resultingprotein, with a 40% of purity, generated in Balb/c mice alymphoproliferative response higher than that induced by the capsidalone and a serotype specific antibodies response higher than thatinduced by PD5.

FIGURES DESCRIPTION

FIG. 1. Cloning strategy of the capsid protein of DEN-2 virus togenerate PDC-2. DEN2 C: Fragment of the capsid protein of DEN-2.

FIG. 2. Analysis by SDS-PAGE at 15% of the PDC-2 semipurificationprocess. 1. Rupture supernatant. 2 and 3. Fraction not adsorbed to QSepharose FF. 4. Fraction eluted with NaCl 1M.

FIG. 3. Analysis by SDS-PAGE at 15% of the PDC-2 purificationprocess. 1. Rupture supernatant, 2. Fraction not absorbed to the gel, 3.Washed (350 mM NaCl), 4. Eluted fraction (750 mM NaCl), 5. Fraction inTris 10 mM, EDTA 1 mM.

FIG. 4. Chromatographic profile in Superdex 200 of the semipurified (A)and pure (B) preparations of PDC-2.

FIG. 5. Electronic microscopy pictures of the pure PDC-2 preparationbefore (A) and after (B) the treatment with oligonucleotides.

FIG. 6. Cloning strategy of the capsid protein of DEN-1 virus togenerate PDC-1. DEN1 C: Fragment of the capsid protein of DEN-1.

FIG. 7. Analysis by SDS-PAGE at 15% of the PDC-1 semipurificationprocess. 1. Molecular weight marker. 2. Rupture supernatant 3. Fractionnot adsorbed to Q Sepharose FF.

EXAMPLES Example 1

Cloning and Expression of PDC-2

The nucleotide sequence that codes for amino acids 1 to 99 of the capsidprotein from DEN-2 virus (Sequence No. 3), was amplified with theoligonucleotides identified in the sequence list as Sequence No. 1 andSequence No. 2 from the DEN-2 virus strain genotype Jamaica (Deubel V.,Kinney R. M., Trent D. W. Nucleotide sequence and deduced amino acidsequence of the nonstructural proteins of Dengue type 2 virus, Jamaicagenotype: Comparative analysis of the full-length genome. Virology1988.165:234-244).

The vector was created by digestion of the plasmid pQE-30 withBamHI/HindIII, which contains the phage 15 promoter and a 6-histidinetail in the N-terminal region (Sequence No. 6). Upon ligation, thepotential recombinants were analyzed by restriction enzyme digestion andpositive clones were sequenced to check up the junctions.

Competent cells XL-1 Blue (Hanahan D. 1983. Studies on transformation ofEscherichia coli with plasmids. J. Mol. Biol. 166:557-580) weretransformed with the selected clone called pDC-2 (FIG. 1 and SequenceNo. 4). The transformed E. coli strains were cultivated in Luria Bertanimedium (LB) supplemented with Ampicilline 50 μg/mL for 10 h at 37° C.Isopropyl-B-D-thiogalactopyranoside (IPTG) to a final concentration of 1mM was used for the induction of the promoter. Upon growing the colony,an SDS-PAGE of the cellular lysate was done. As a result, a 15-kDA bandwas obtained. The protein was recognized by an anti-DEN-2 hyperimmuneascitic fluid (HMAF). This protein was denominated PDC-2 (Sequence No.5).

Example 2 Semipurification and Characterization of PDC-2

The biomass obtained from the E. coli strain transformed with pDC-2 andgrown at 37° C. was disrupted by French press. The recombinant proteinwas obtained equally distributed between the soluble and insolublefractions. The soluble fraction was subjected to an anionic interchangechromatography, using a Q Sepharose FF column and the buffer Tris 10 mMpH 8. The protein in the non-absorbed fraction was obtained with 40%purity and was used for the immunological studies (FIG. 2).

Example 3 Immunological Evaluation of Semipurified PDC-2

Three groups of 30 Balb/c mice were used. Two of them were immunizedwith 10 ug of the recombinant protein by intraperitoneal route, usingFreund's Adjuvant (FA) in one of the groups and aluminum hydroxide inthe other. The soluble fraction resulting from the rupture of thepQE-30-transformed cells was used as negative control adjuvanted withFA; 10 animals were bled 15 days after the third dose and the antibodytiters against DEN-2 were determined by ELISA. After the immunizationwith the recombinant protein, formulated in either adjuvant, no antibodytiters were obtained.

Table 1. Antibody titers against DEN-2 from the sera obtained uponimmunization of mice with semipurified PDC-2.

TABLE 1 Antibody titers against DEN-2 from the sera obtained uponimmunization of mice with semipurified PDC-2. ELISA Titers against DEN-2PDC-2 PDC-2 XL-1 Blue Mouse Freund A. Aluminum hydroxide Freund (Neg.Control) 1 <1:100 <1:100 <1:100 2 <1:100 <1:100 <1:100 3 <1:100 <1:100<1:100 4 <1:100 <1:100 <1:100 5 <1:100 <1:100 <1:100 6 <1:100 <1:100<1:100 7 <1:100 <1:100 <1:100 8 <1:100 <1:100 <1:100 9 <1:100 <1:100<1:100 10 <1:100 <1:100 <1:100

Example 4 Protection Assay

For the evaluation of the protection conferred to mice against challengewith lethal homologous DEN virus by the immunization with the describedvariants, 10 mice were used from each of the groups immunized with therecombinant protein adsorbed in aluminum hydroxide and with the controlpreparation. Each animal received a dose of 100 LD₅₀ of lethal DEN-2virus by intracranial inoculation and was observed for 21 days to obtainthe percentages of lethality in terms of death by viral encephalitis. Asa positive control, a group of 10 mice immunized with infective DEN-2virus (10⁴ pfu) was used. All mice in the positive control groupsurvived, while in the negative control group all mice were sick by day7-11 after challenge and 100% mortality was obtained by day 21. Finally,the group immunized with the recombinant protein PDC-2 presented 44.4%protection (table 2).

TABLE 2 Percentage of survival in PDC-2 immunized mice upon challengewith the homologous lethal Dengue virus. Survival Immunogen percentageXL-1 blue 0 DEN-2 100 PDC-2 alum. 44.4 * It was calculated: (# desurvivors)/(total # of mice). Data of survivors were taken 21 days afterchallenge.

Example 5 Lymphoproliferative Response

The rest of the animals from the group immunized with the capsid proteinadjuvanted with aluminum hydroxide were sacrificed 30 days after thelast dose. Then, their spleens were extracted and thelymphoproliferative response to DEN-2 was studied. The results in table3 show the stimulation indexes obtained.

TABLE 3 Stimulation indexes against the homologous serotype of thelymphocytes from immunized mice. PDC-2 Aluminum hydroxide DEN-2** 8*Control 1.5 Antigen*** PHA**** 7 *Stimulation index: quotient average ofcounts/minutes of samples between counts/minutes of the ADN spontaneoussynthesis control. **Preparation of DEN-2 infected mice brain.***Preparation of not infected mice brain. ****PhytohemaglutininaMitogen (Positive Control).

Example 6 Purification of PDC-2

The biomass obtained from the E. coli strain transformed with pDC-2 andgrown at 37° C. was disrupted by French press. The recombinant proteinwas obtained equally distributed between the soluble and insolublefractions. The soluble fraction was subjected to a cationic interchangechromatography, using an SP-Sepharose FF column and the buffer Tris 10mM, Tween 0.5%, urea 7M, pH 8. The column was washed with bufferdiethanolamine 30 mM, NaCl 350 mM, pH 10.3. The elution of the proteinof interest was done with buffer diethanolamine 30 mM, NaCl 750 mM, pH10.3. Once eluted the protein, the buffer was exchanged using G-25columns. Finally, the protein was obtained with 96% purity in bufferTris 10 mM, EDTA 1 mM (FIG. 3).

Example 7 Characterization of the Semipurified and Purified Variants

With the aim of characterizing the state of aggregation of thesemipurified and the purified preparations, gel filtrationchromatographies were done using the TSK-5000 column (Tosoh bioscience,Japan). After applying the semipurified sample, a homogeneous and majorpeak was obtained, with a retention time ranging from 15 to 20 minutes,evidencing the presence of high molecular weight species (FIG. 4A).Contrarily, in the sample from the highly purified fraction of thecapsid protein, retention times of 30 minutes were detected,corresponding to the dimeric form of the molecule (FIG. 4B).

Example 8 Studies of Reparticulation “In Vitro”

In order to reparticulate the pure capsid protein in a dimeric form, thebuffer was exchanged to Hepes 25 mM, KAc 100 mM, MgAc2 1.7 mM, pH 7.4.After heating the protein and the mixture of oligonucleotides for 1 minat 37° C., they were incubated in an equal volume for 30 min at 30° C.As a negative control of the experiment, the protein was incubatedwithout the oligonucleotides. When both preparations were observed withan electron microscope, a large quantity of particles of approximately21 nm diameter, were observed in the sample of protein previouslyincubated with the mixture of oligonucleotides, while in the controlsample no particles were observed (FIG. 5).

Example 9 Immunological Evaluation in Mice of the Purified Capsid

Five groups of 20 Balb/c mice were used. Two of them were immunized with10 ug of the dimeric purified recombinant protein by intraperitonealroute, using aluminum hydroxide and Freund's adjuvant. Another group wasimmunized with 10 ug of the purified and particulated capsid proteinadjuvanted with aluminum hydroxide. The soluble fraction from therupture of XL-1 blue cells transformed with the plasmid pQE-30 andsubjected to the same purification steps than PDC-2 was used as negativecontrol, adjuvanted with Freund's adjuvant. The fifth group wasimmunized with DEN-2 virus as positive control. One month after the lastdose 10 animals from each group received a dose of 100 LD₅₀ of lethalDEN-2 by intracranial inoculation and were observed for 21 days toobtain the percentages of survival. All mice in the positive controlgroup survived, while in the negative control group all mice were sickby day 7-11 after challenge and 0% mortality was obtained. Finally, fromthe groups immunized with the recombinant protein, the group immunizedwith pure dimeric PDC-2 presented a 20% protection when immunized withaluminum hydroxide and a 40% protection when Freund's adjuvant was used.Additionally, in the group that received the reparticulated pure proteinadjuvanted with aluminum hydroxide. 90% of mice were protected (Table4).

TABLE 4 Percentage of survival in mice immunized with the proteinvariants assayed upon challenge with the homologous lethal Dengue virus.Immunogen (adjuvant) Survival percentage* Xl-1 Blue (Freund) 0 Pure anddimeric PDC-2 (Aluminum) 20 Pure and dimeric PDC-2 (Freund) 40 Pure andreparticulated 90 PDC-2 (Aluminum) DEN-2 100 *It was calculated: (# desurvivors)/(total # of mice). Data of survivors were taken 21 days afterchallenge.

Example 10 Lymphoproliferative Response

The rest of the animals from the groups immunized with the capsidprotein (10 animals), either dimeric or reparticulated, adjuvanted withaluminum hydroxide, were sacrificed 15 days after the last dose. Then,their spleens were extracted and the lymphoproliferative response toDEN-2 was studied. The results in table 5 show the stimulation indexesobtained.

TABLE 5 Stimulation indexes against the homologous serotype of thelymphocytes from immunized mice. Pure and reparticulated Pure PDC-2PDC-2 DEN-2** 10* 4 Antigen 1.5 1.2 Control*** PHA**** 7 8 *Stimulationindex: quotient average of counts/minutes of samples betweencounts/minutes of the ADN spontaneous synthesis control. **Preparationof DEN-2 infected mice brain. ***Preparation of not infected mice brain.****Phytohemaglutinina Mitogen (Positive Control).

Example 11 Immunological Evaluation of the Mixture Formed by PD5 andPDC-2

Twenty animals were inoculated with the mixture of 10 ug of theparticulated pure capsid protein and 20 ug of protein PD5 (Sequence No.23) in three doses spaced fifteen days apart. A group immunized with 10ug of the pure capsid protein, a group immunized with 20 ug of proteinPD5 mixed with the equivalent volume of PDC-2 but obtained from anegative control run, and a group immunized with protein P64k, thecarrier protein present in the construction of PD5, were used ascontrols. In all cases, aluminum hydroxide was used as adjuvant.

Fifteen days after the last dose, the animals were bled and the seratested for antiviral antibodies by ELISA. As shown in tables 6 and 7,the group immunized with the mixture developed serotype-specificantibodies with titers higher than those of the group immunized onlywith protein PD5 and, at the same time, titers in these two groups werehigher than those in the group immunized with protein PDC-2, where noAbs against DEN-2 virus were detected. On the other hand, 10 additionalanimals were taken from each group for lymphoproliferation assays. Thecells from the spleens of these animals were extracted and stimulatedwith the infective DEN-2 virus. As shown in table 8, in the groupimmunized with the mixture the stimulation indexes were higher thanthose in the group immunized with the capsid protein only. The loweststimulation indexes were obtained in the group immunized with proteinPD5.

TABLE 6 Antibody titers against DEN-2 virus in sera obtained after theimmunization. Groups inoculated with: Mouse PDC-2 PDC-2/PD5 PD5 P64k 1<1:100 <1:128000 <1:64000 <1:100 2 <1:100 <1:320000 <1:32000 <1:100 3<1:100 <1:320000 <1:64000 <1:100 4 <1:100 <1:320000 <1:16000 <1:100 5<1:100 <1:64000  <1:64000 <1:100 6 <1:100 <1:128000  <1:128000 <1:100 7<1:100 <1:64000  <1:64000 <1:100 8 <1:100 <1:128000 <1:32000 <1:100 9<1:100 <1:320000 <1:64000 <1:100 10 <1:100 <1:320000 <1:32000 <1:100

TABLE 7 Determination of the serotype-specificity of the antibodiescontained in the mixtures of the sera obtained from each group. ViralGroups inoculated with: Antigen PDC-2 PDC-2/PD5 PD5 P64k DEN-1 <1:100<1:200 <1:200 <1:100 DEN-2 <1:100    1:320 000   1:64000 <1:100 DEN-3<1:100 <1:200 <1:200 <1:100 DEN-4 <1:100 <1:200 <1:200 <1:100

TABLE 8 Stimulation indexes against the homologous serotype of thelymphocytes from immunized mice. PDC- PDC-2 2/PD5 PD5 P64k DEN-2** 9* 112.1 1.1 Antigen*** 1.3 1.6 1.5 1.2 Control (−) PHA**** 7.5 7.3 7.9 8*Stimulation index: quotient average of counts/minutes of samplesbetween counts/minutes of the ADN spontaneous synthesis control.**Preparation of DEN-2 infected mice brain. ***Preparation of notinfected mice brain. ****Phytohemaglutinina Mitogen (Positive Control).

Example 12 CD8 Depletion Studies

The reparticulated and the dimeric capsid proteins were inoculated inBalb/c mice to obtain some evidence of induction of cellular immuneresponse. A preparation obtained from cells transformed with the plasmidused to generate pDC-2, and by a purification process similar to the oneused for the protein PDC-2, was employed as a negative control.

Three doses of the protein (20 ug) were administered to groups of 20animals, using aluminum hydroxide as adjuvant. One month after the lastdose, 1 mg of a rat anti-mouse CD8 mAb, able to deplete the cells of themouse immune system containing this marker was administered to half ofthe animals of each group. On the next day, all the animals werechallenged with 100 LD₅₀ (Median Lethal doses) of DEN-2 virus. They wereobserved for the onset of signs of disease and deaths were recorded.

In the case of the immunized non-treated groups, 20 and 80% protectionwas obtained in the groups immunized with the dimeric and thereparticulated capsid, respectively. Parallely, in the treated groupsthe percentage of protection was lower than in the non-treated groups:0% protection for the dimeric PDC-2 and 10% protection for thereparticulated protein. In the case of the negative control group noprotection was obtained in either the treated or the non-treatedanimals.

TABLE 9 Challenge assay with DEN-2 lethal virus in the animals immunizedwith variants of the recombinant capsid *Survival percentages Survivalpercentages in mice treated in mice non with the treated with the Groupsanti-CD8 mAb anti-CD8 mAb PCD12 reparticulated 10 80 PCD12 0 20non-particulated Control (−) 0 0 *It was calculated: (# desurvivors)/(total # of mice). Dataa of survivors were taken 21 daysafter challenge.

Example 13 Obtaining and Semipurification of the DEN-1 Protein

The nucleotide sequence that codes for amino acids 1 to 100 of thecapsid protein of DEN-1 virus (Sequence No. 7), was amplified with theoligonucleotides identified in the sequence list as Sequence No. 8 andSequence No. 10 from the DEN-1 viral strain. The vector was generated bydigestion BamHI/HindIII of the plasmid pQE-30, which contains the phageT5 promoter and a 6 histidine tail in the N-terminal region (SequenceNo. 6). Upon ligation, the recombinants were analyzed by restriction andthe positives clones were sequenced to check the junctions. Competentcells XL-1 Blue (Hanahan D. 1983. Studies on transformation ofEscherichia coli with plasmids. J. Mol. Biol. 166:557-580) weretransformed with the selected clone called pDC-1 (FIG. 6 y Sequence No.10). The E. coli strains transformed were cultivated in LB supplementedwith Ampicilline 50 μg/mL for 10 h at 37° C.Isopropyl-B-D-thiogalactopyranoside (IPTG) to a final concentration of 1mM was used for the induction of the promoter. Upon growing the colony,an SDS-PAGE of the cellular lysate was done. As a result a 15-kDA bandwas obtained. The protein was recognized by an anti-DEN-1 HMAF. Thisprotein was denominated PDC-1 (Sequence No. 11).

Example 14 Semipurification and Characterization of PDC-1

The biomass obtained from the E. coli strain transformed with pDC-1 andgrown at 37° C. was disrupted by French press. The recombinant proteinwas obtained equally distributed between the soluble and insolublefractions. From the soluble fraction an anionic interchangechromatography was done, using a Q Sepharose FF column and the bufferTris 10 mM pH 8. The protein in the non-absorbed fraction was obtainedwith 45% of purity, and was used to the immunological studies.

Example 15 Immunological Evaluation of Semipurified PDC-1

Two groups of 30 Balb/c mice were used. One of them was immunized with10 ug of the recombinant protein by intraperitoneal route, using thealuminum hydroxide as adjuvant. The soluble fraction resulting from therupture of the pQE-30-transformed cells adjuvanted with aluminumhydroxide was used as negative control. A part of the animals (10 mice)were bled 15 days after the third dose and the antibody titers againstDEN-1 were determined by ELISA. After the immunization with therecombinant protein, no antiviral antibody titers were obtained.

TABLE 10 Antibodies titers against DEN-1 virus from sera obtained afterthe immunization with the semipurified PDC-1. Anti- DEN-1 ELISA titersXL-1 blue Mouse Control (−) PDC-1 1 <1:100 <1:100 2 <1:100 <1:100 3<1:100 <1:100 4 <1:100 <1:100 5 <1:100 <1:100 6 <1:100 <1:100 7 <1:100<1:100 8 <1:100 <1:100 9 <1:100 <1:100 10 <1:100 <1:100

Example 16 Protection Assay

For the evaluation of the protection conferred to mice against challengewith lethal homologous DEN virus by the immunization with the describedvariants, 10 mice were used from each of the groups immunized with therecombinant protein adsorbed in aluminum hydroxide and with the controlpreparation. Each animal received a dose of 100 LD₅₀ of lethal DEN-1 byintracranial inoculation and was observed for 21 days to obtain thepercentages of lethality in terms of death by viral encephalitis. As apositive control, a group of 10 mice immunized with infective DEN-1virus (10⁴ pfu) was used. All mice in the positive control groupsurvived, while in the negative control group all mice were sick at day7-11 after challenge and 100% mortality was obtained at day 21. Finally,the group immunized with the recombinant protein PDC-1 presented 50% ofprotection (Table 11).

TABLE 11 Percentage of survival in mice immunized with the proteinvariants assayed upon challenge with the homologous lethal DEN virus.Survival Immunogen percentages* XL-1 blue 0 (Control −) DEN-1 100(Control +) PDC-1 50 *It was calculated: (# de survivors)/(total # ofmice). Data of survivors were taken 21 days after challenge.

Example 17 Lymphoproliferative Response

The rest of the animals of the group immunized with the protein PDC-1were sacrificed 15 days after the last dose. Then, their spleens wereextracted and the lymphoproliferative response to DEN-1 was studied. Theresults in table 12 show the stimulation indexes obtained.

TABLE 12 Stimulation indexes against the homologous serotype of thelymphocytes from immunized mice. PDC-1 aluminum hydroxide DEN1** 8*Control 1.5 Antigen*** PHA**** 7 *Stimulation index: quotient average ofcounts/minutes of samples between counts/minutes of the ADN spontaneoussynthesis control. **Preparation of DEN-2 infected mice brain.***Preparation of not infected mice brain. ****PhytohemaglutininaMitogen (Positive Control).

Example 18 Cloning and Expression of PDC-2 DomIII

The nucleotide sequence that codes for amino acids 286 to 426 of theenvelope protein from DEN-2 (Sequence No. 12), corresponding to theregion of the domain 111 of the protein, was amplified with theoligonucleotides identified in the sequence list as Sequence No. 13 andSequence No. 14 from the DEN-2 virus strain genotype Jamaica (Deubel V.,Kinney R. M., Trent D. W. Nucleotide sequence and deduced amino acidsequence of the nonstructural proteins of Dengue type 2 virus, Jamaicagenotype: Comparative analysis of the full-length genome. Virology1988.165:234-244).

The vector was created by digestion of the plasmid pDC-2 withBamHI/BamHI, which contains the phage T5 promoter, a 6-histidine tail inthe N-terminal region and the region corresponding to 100 amino acids ofthe capsid protein of DEN-2 virus. Upon ligation, the potentialrecombinants were analyzed by restriction enzyme digestion and positiveclones were sequenced to check up the junctions. Finally the cloneselected was named pDC-2 Dom III (Sequence No 15).

Competent cells XL-1 Blue (Hanahan D. 1983. Studies on transformation ofEscherichia coli with plasmids. J. Mol. Biol. 166:557-580) weretransformed with the selected clone called pDC-2 DomIII. The E. colistrains transformed were cultivated in LB supplemented with Ampicilline50 μg/mL for 10 h at 37° C. isopropyl-B-D-thiogalactopyranoside (IPTG)to a final concentration of 1 mM was used to the induction of thepromoter. Upon growing the colony, an SDS-PAGE of the cellular lysatewas done. As a result, a 30-kDA band was obtained. The protein wasrecognized by an anti-DEN-2 HMAF. This protein was denominated PDC-2 DomIII (Sequence No. 16).

Example 19 Semipurification and Characterization of PDC-2 Dom III

The biomass obtained from the E. coli strain transformed with pDC-2DomIII and grown at 37° C. was disrupted by French press. Therecombinant protein was obtained equally distributed between the solubleand insoluble fractions. From the soluble fraction an anionicinterchange chromatography was done, using a Q Sepharose FF column andthe buffer Tris 10 mM pH 8. The protein in the non-absorbed fraction wasobtained with 40% of purity, and was used to the immunological studies(FIG. 2).

Example 20 Immunological Evaluation in Mice of the Semipurified PDC-2Dom III

Five groups of 30 Balb/c mice were used. One of the groups was immunizedwith 10 ug of the recombinant protein by intraperitoneal route, usingaluminum hydroxide as adjuvant. The soluble fraction resulting from therupture of the XL-1 Blue cells transformed with the plasmid pQE-30 wasused as negative control, adjuvanted with aluminum hydroxide. Anothertwo groups were included as controls. One of them was immunized with theprotein PDC-2 and the other with the protein PD5 (this protein containsthe domain III region of the envelope protein of DEN-2 virus). Tenanimals from each group were bled 15 days after the third dose and theantibody titers against DEN-2 were determined by ELISA. As shown inTables 13 and 14, the group immunized with PDC-2 Dom III developed hightiters of serotype-specific antibodies against DEN-2, higher than thoseinduced by the protein PD5. These results demonstrate that the geneticcombination with the capsid protein enhances the antiviral immuneresponse elicited by the domain III of the envelope protein.

TABLE 13 Antibodies titers against DEN-2 virus from sera obtained afterthe immunization with the Dom III-capsid protein. Groups immunized with:Mouse PDC-2 PDC-2 Dom III PD5 P64k 1 <1:100 <1:320000 <1:32000 <1:100 2<1:100 <1:640000 <1:32000 <1:100 3 <1:100 <1:640000 <1:64000 <1:100 4<1:100 <1:640000 <1:64000 <1:100 5 <1:100 <1:128000 <1:64000 <1:100 6<1:100 <1:320000 <1:32000 <1:100 7 <1:100 <1:128000 <1:64000 <1:100 8<1:100 <1:64000   <1:128000 <1:100 9 <1:100 <1:64000  <1:32000 <1:100 10<1:100 <1:128000 <1:64000 <1:100

TABLE 14 Determination of the serotype-specificity of the antibodiescontained in the mixtures of the sera obtained from each group. ViralGroups inoculated with: Antigen PDC-2 PDC-2 Dom III PD5 P64k DEN-1<1:100 <1:200 <1:200 <1:100 DEN-2 <1:100    1:320 000    1:64 000 <1:100DEN-3 <1:100 <1:200 <1:200 <1:100 DEN-4 <1:100 <1:200 <1:200 <1:100

On the other hand, 10 additional animals were taken from each group forthe lymphoproliferation assays. The cells from the spleens of theseanimals were extracted and stimulated with the infective DEN-2 virus.Table 15 shows that in the group immunized with the combination, thestimulation indexes were higher than those in the group immunized withthe capsid protein only. The stimulation indexes in the group immunizedwith protein PD5 were the lowest.

TABLE 15 Stimulation indexes against the homologous serotype of thelymphocytes from immunized mice. PDC-2 PDC-2 Dom III PD5 P64k DEN-2**9.5* 11.6 2.2 1.2 Antigen*** 1.2 1.1 1.2 1.6 Control (−) PHA**** 7.6 7.47.5 7.9 *Stimulation index: quotient average of counts/minutes ofsamples between counts/minutes of the ADN spontaneous synthesis control.**Preparation of DEN-2 infected mice brain. ***Preparation of notinfected mice brain. ****Phytohemaglutinina Mitogen (Positive Control).

Incorporation of Sequence Listing

Incorporated herein by reference in its entirety is the Sequence Listingfor the application. The Sequence Listing is disclosed on acomputer-readable ASCII text file titled, “sequence_listing.txt”,created on Jul. 23, 2008. The sequence_listing.txt file is 44 kb insize.

1. A pharmaceutical composition capable of generating an immune responseagainst Dengue virus, comprising a capsid protein of Dengue virus type1, a capsid protein of Dengue virus type 2, a capsid protein of Denguevirus type 3, and a capsid protein of Dengue virus type 4, wherein thecapsid protein of Dengue serotype 1, the capsid protein of Dengueserotype 2, the capsid protein of Dengue serotype 3, or the capsidprotein of Dengue serotype 4 is fused with a capsid protein of Denguevirus type 1, a capsid protein of Dengue virus type 2, a capsid proteinof Dengue virus type 3, or a capsid protein of Dengue virus type
 4. 2.The pharmaceutical composition of claim 1, wherein the capsid protein ofDengue serotype 1, the capsid protein of Dengue serotype 2, the capsidprotein of Dengue serotype 3, or the capsid protein of Dengue serotype 4is fused to an immunogenic antigen.
 3. The pharmaceutical composition ofclaim 2, wherein the immunogenic antigen is a peptide having the aminoacid sequence comprising SEQ ID NO:
 19. 4. The pharmaceuticalcomposition of claim 1, further comprising a capsid protein of Denguevirus type 1, a capsid protein of Dengue virus type 2, a capsid proteinof Dengue virus type 3, and a capsid protein of Dengue virus type 4, ora combination thereof.
 5. The pharmaceutical composition of claim 1,further comprising an immunogenic antigen.
 6. A pharmaceuticalcomposition capable of generating an immune response against Denguevirus, comprising a) a capsid protein of Dengue virus type 1, a capsidprotein of Dengue virus type 2, a capsid protein of Dengue virus type 3,and a capsid protein of Dengue virus type 4: and b) an immunogenicantigen wherein the immunogenic antigen is a peptide having the aminoacid sequence comprising SEQ ID NO:
 22. 7. A pharmaceutical compositioncapable of generating an immune response against Dengue virus,comprising a capsid protein of Dengue virus type 1, a capsid protein ofDengue virus type 2, a capsid protein of Dengue virus type 3, and acapsid protein of Dengue virus type 4, wherein the capsid protein is inaggregated form or particulate form.
 8. The pharmaceutical compositionof claim 1, further comprising a pharmacologically acceptable vehicleand an adjuvant.
 9. The pharmaceutical composition of claim 1, whereinthe pharmaceutical composition is suitable for oral, intramuscular,subcutaneous, mucosal, or intravenous administration.
 10. Apharmaceutical composition capable of generating an immune responseagainst Dengue virus, comprising a capsid protein of Dengue virus type1, a capsid protein of Dengue virus type 2, a capsid protein of Denguevirus type 3, and a capsid protein of Dengue virus type 4, wherein thecapsid protein of Dengue serotype 1, the capsid protein of Dengueserotype 2, the capsid protein of Dengue serotype 3, or the capsidprotein of Dengue serotype 4 is fused to a peptide having the amino acidsequence consisting of SEQ ID NO: 19.